Consequences of primer binding-sites polymorphisms on genotyping practice

Abstract

Herein we investigated the effect of primer binding site polymorphisms in achieving correct genotyping when a mismatch occurs in distinct positions of the primer sequence. For that purpose primer sequences were designed in order to carry either allelic form at the 3’ end and at 3 bp, 5 bp and 7 bp apart from the 3’end of an intronic polymorphism (rs2247836) ob- served in phenylalanine hydroxylase (PAH) gene. For one of the alleles annealing failure was obtained when the mismatch occurs at all the four primer-site loca- tions. Primer sequences carrying the alternative SNP allele resulted to be less specific as the distance to the primer-3’ end was increased. Altogether, these results revealthat effects in the extension of the annealing failure is allele and mismatch-position dependent.